The overall goals of this proposal are to characterize the lineal origins of blood cells and to determine the effects of cell interactions upon fate decisions made by vertebrate blood cell precursors. Our identification of separate sites of production of macrophages and red blood cells in the zebrafish will allow for a dissection of the different environmental and genetic influences that cause precursors to adopt myeloid and erythroid fates. We have identified a key interaction with the paraxial mesoderm that is required for proper RBC formation and will further study the implications of this interaction for future diagnostic and therapeutical implications related to leukemias, myelomas, lymphomas, myelodysplastic syndromes and anemias. An understanding of the influences that affect cell fate choices will potentially be important in the screening, culturing and in vitro differentiation of stem cells for such procedures as bone marrow transplants and the treatment of genetic disorders such as thalassemia and sickle cell anemia. Our general strategy is to use the genetic and cellular advantages of the model system zebrafish to understand blood cell differentiation. The zebrafish has become a viable and important model system for blood cell differentiation in amniotes, including humans. There are currently over 50 described zebrafish mutations in 26 complementation groups that exhibit blood defects. Characterization and cloning of such hematopoietic mutants has made substantial contributions to human disease research, for instance the zebrafish mutant, sauternes, currently serves as the only model of human congenital sideroblastic anemia. Transgenic lines of zebrafish serve as model systems for cancer studies, for instance, lines of zebrafish expressing c-myc exhibit symptoms of T-cell acute leukemia. The zebrafish mutant spadetail fails to form early Red Blood Cell lineages but retains the ability to form early macrophage lineages. We propose to study the role of both cell-autonomous and environmental factors that are required to form the erythroid and myeloid cell fate by comparing the process of blood formation between wild-type and spadetail mutant zebrafish. Specifically, we will perform fate map studies to which the zebrafish is particularly amenable, for the purpose of directly determining the origins of myeloid and red blood cell precursors. We will determine when the spadetail gene function and environmental signals are required to cooperate for proper red blood cell production by transplanting wildtype cells into various positions within the spadetail mutant to rescue the loss of red blood cell phenotype. We will also use cell transplantations to determine if an interaction with paraxial mesoderm can respecify myeloid precursors to adopt a red blood cell fate.